Prem K. Premsrirut

Control of the senescence-associated secretory phenotype by NF-κB promotes senescence and enhances chemosensitivity

Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.

A pipeline for the generation of shRNA transgenic mice

RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches—inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.

Reversible suppression of an essential gene in adult mice using transgenic RNA interference

RNAi has revolutionized loss-of-function genetics by enabling sequence-specific suppression of virtually any gene. Furthermore, tetracycline response elements (TRE) can drive expression of short hairpin RNAs (shRNAs) for inducible and reversible target gene suppression. Here, we demonstrate the feasibility of transgenic inducible RNAi for suppression of essential genes. We set out to directly target cell proliferation by screening an RNAi library against DNA replication factors and identified multiple shRNAs against Replication Protein A, subunit 3 (RPA3). We generated transgenic mice with TRE-driven Rpa3 shRNAs whose expression enforced a reversible cell cycle arrest. In adult mice, the block in cell proliferation caused rapid atrophy of the intestinal epithelium which led to weight loss and lethality within 8–11 d of shRNA induction. Upon shRNA withdrawal, villus atrophy and weight loss were fully reversible. Thus, shRpa3 transgenic mice provide an interesting tool to study tissue maintenance and regeneration. Overall, we have established a robust system that serves the purpose of temperature-sensitive alleles in other model organisms, enabling inducible and reversible suppression of essential genes in a mammalian system.

PTEN action in leukaemia dictated by the tissue microenvironment

PTEN encodes a lipid phosphatase that is underexpressed in many cancers owing to deletions, mutations or gene silencing. PTEN dephosphorylates phosphatidylinositol (3,4,5)-triphosphate, thereby opposing the activity of class I phosphatidylinositol 3-kinases that mediate growth- and survival-factor signalling through phosphatidylinositol 3-kinase effectors such as AKT and mTOR. To determine whether continued PTEN inactivation is required to maintain malignancy, here we generate an RNA interference-based transgenic mouse model that allows tetracycline-dependent regulation of PTEN in a time- and tissue-specific manner. Postnatal Pten knockdown in the haematopoietic compartment produced highly disseminated T-cell acute lymphoblastic leukaemia. Notably, reactivation of PTEN mainly reduced T-cell leukaemia dissemination but had little effect on tumour load in haematopoietic organs. Leukaemia infiltration into the intestine was dependent on CCR9 G-protein-coupled receptor signalling, which was amplified by PTEN loss. Our results suggest that in the absence of PTEN, G-protein-coupled receptors may have an unanticipated role in driving tumour growth and invasion in an unsupportive environment. They further reveal that the role of PTEN loss in tumour maintenance is not invariant and can be influenced by the tissue microenvironment, thereby producing a form of intratumoral heterogeneity that is independent of cancer genotype.

Prediction of potent shRNAs with a sequential classification algorithm

We present SplashRNA, a sequential classifier to predict potent microRNA-based short hairpin RNAs (shRNAs). Trained on published and novel data sets, SplashRNA outperforms previous algorithms and reliably predicts the most efficient shRNAs for a given gene. Combined with an optimized miR-E backbone, >90% of high-scoring SplashRNA predictions trigger >85% protein knockdown when expressed from a single genomic integration. SplashRNA can significantly improve the accuracy of loss-of-function genetics studies and facilitates the generation of compact shRNA libraries.

Transgenic, inducible RNAi in megakaryocytes and platelets in mice

Summary  Background: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)‐regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. Objective: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. Methods and results: Using a tet‐regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet‐on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV‐rtTA mice) display inducible reporter gene expression with unusual and near‐complete efficiency in megakaryocytes and platelets. To test whether the CMV‐rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet‐regulated short hairpin RNA (shRNA) targeting Bcl‐xL, a pro‐survival Bcl‐2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl‐xL in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl‐xL levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl‐xL, clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. Conclusions: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.

Creating Transgenic shRNA Mice by Recombinase-Mediated Cassette Exchange

RNA interference (RNAi) enables sequence-specific, experimentally induced silencing of virtually any gene by tapping into innate regulatory mechanisms that are conserved among most eukaryotes. The principles that enable transgenic RNAi in cell lines can also be used to create transgenic animals, which express short-hairpin RNAs (shRNAs) in a regulated or tissue-specific fashion. However, RNAi in transgenic animals is somewhat more challenging than RNAi in cultured cells. The activities of promoters that are commonly used for shRNA expression in cell culture can vary enormously in different tissues, and founder lines also typically vary in transgene expression due to the effects of their single integration sites. There are many ways to produce mice carrying shRNA transgenes and the method described here uses recombinase-mediated cassette exchange (RMCE). RMCE permits insertion of the shRNA transgene into a well-characterized locus that gives reproducible and predictable expression in each founder and enhances the probability of potent expression in many cell types. This procedure is more involved and complex than simple pronuclear injection, but if even a few shRNA mice are envisioned, for example, to probe the functions of several genes, the effort of setting up the processes outlined below are well worthwhile. Note that when creating a transgenic mouse, one should take care to use the most potent shRNA possible. As a rule of thumb, the sequence chosen should provide >90% knockdown when introduced into cultured cells at single copy (e.g., on retroviral infection at a multiplicity of ≤0.3).

Abstract B37: RNAi and CRISPR/Cas9-based in vivo models for drug discovery

With the advent of CRISPR-Cas9 technology, the speed and precision in which genetically engineered mouse models can be created is unprecedented. We now have at our disposal a genetic toolbox that will enable the rapid generation of sophisticated mouse models of cancer. Recently, an inducible CRISPR-Cas9 (iCRISPR) system was described that enables doxycycline-regulated Cas9 induction of widespread gene mutagenesis in multiple tissues. Previously, we also demonstrated how inducible RNA interference (RNAi) can be exploited experimentally to effectively and reversibly silence nearly any gene target not only in vitro but also in live mice. Here, we take advantage of these powerful technologies and combine both tet-inducible CRISPR-Cas9 and inducible RNAi-mediated gene silencing to develop animal models in which both de novo tumorigenesis can be induced by Cas9-mediated genome editing and therapeutic strategies assessed downstream via RNA interference-mediated gene silencing. By using this combination of CRISPR/Cas9 and RNAi technologies, we are able to not only model disease pathogenesis, but also mimic drug therapy in the same mice, giving us exceptional capabilities to perform preclinical studies in vivo. Using our robust flexible system, we have created a cost-effective and scalable platform for the production of complex genetically engineered mouse models of cancer with RNAi silencing of nearly any gene - mice with enormous predictive power that will shape our development of better tolerated therapies. Citation Format: Prem K. Premsrirut, Gregory Martin, Lukas Dow, Sang Yong Kim, Johannes Zuber, Scott Lowe, Greg Hannon. RNAi and CRISPR/Cas9 based in vivo models for drug discovery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4188.

Abstract LB-335: Induction of Ribosomal Checkpoint Induced Senescence (RCIS) for the treatment of liver cancer

While ribosomal proteins are generally considered to be essential genes, the existence of ribosomopathies suggests that a therapeutic window for exploiting ribosomal proteins as targets for cancer therapy may exist. We report distinct stress responses of tumor cells and normal cells upon knockdown of Rpl15, a ribosomal protein which was identified in a direct in vivo shRNA screen for new therapeutic targets in liver cancer. While normal cells undergo a reversible cell cycle arrest, we found that in tumor cells, the knockdown of Rpl15 triggers a ribosomal stress response followed by induction of cellular senescence, designated ribosomal checkpoint induced senescence (RCIS). Importantly, Rpl15 suppression triggered RCIS independent of any reduction in global protein translation. Using well established therapy resistant murine HCC models, we show that shRNAs targeting Rpl15 potently suppress tumor development in genetically diverse murine HCCs. Using Rpl15 shRNA transgenic mice, allowing for ubiquitous shRNA mediated suppression of Rpl15, we show that systemic Rpl15 suppression can be tolerated for up to 5 days, thus revealing a therapeutic window for metronomic Rpl15 inhibitory therapies. However, molecular modeling analyses revealed that Rpl15 is not druggable by small molecule inhibitors and we thus set out to explore whether RCIS can be induced via interference with other ribosomal proteins or other factors involved in ribosome biogenesis. We generated and screened a focused shRNA library targeting 41 ribosomal proteins and 19 ribosome biogenesis factors and found that apart from Rpl15, only a small subset of shRNAs scored. From a druggability point of view it was interesting, that shRNAs targeting components of the RNA polymerase I complex had scored, as recently a pharmacological RNA polymerase I inhibitor became available. Both genetic and pharmacological inhibition of RNA polymerase I induced RCIS and mediated an excellent prolongation of survival (far superior to the clinically used standard therapy Sorafenib), however in contrast to targeting Rpl15, targeting RNA polymerase I failed to achieve full long term tumor suppression. Mechanistically we found a less efficient immune mediated clearance of senescent cells as a possible explanation. In line with the idea that distinct secretory profiles underlie differences in immune mediated clearance of RCIS cells, cytokine arrays revealed distinct secretory profiles of RCIS induced by Rpl15 suppression and RCIS induced via shRNA or pharmacological RNA polymerase I inhibition. Citation Format: Katharina Wolter, Marina Pesic, Sabrina Klotz, Nicolas Herranz, Torsten Wuestefeld, Tae-Won Kang, Marco Seehawer, Rishabh Chawla, Stefan Zwirner, Jonathan Cotton, Benyuan Zhou, Marcel Kruger, Frank Klawonn, Thomas Longerich, Bence Sipos, Bernd Pichler, Jesus Gil, Martin Eilers, Prem K. Premsrirut, Antti Poso, Lars Zender. Induction of Ribosomal Checkpoint Induced Senescence (RCIS) for the treatment of liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-335. doi:10.1158/1538-7445.AM2017-LB-335

Abstract PR07: RNAi mouse models: Revolutionizing drug discovery in vivo

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi tools including RNAi transgenic mice remains a significant limitation. One main hurdle is the identification of potent RNAi triggers, or short hairpin RNAs (shRNAs), that will induce stable and regulated gene silencing. Due to the lack of understanding of the requirements for shRNA biogenesis and target suppression, many predicted shRNAs fail to efficiently induce gene suppression. We have developed a “Sensor assay” that enables the biological identification of effective shRNAs at large scale and show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. In addition, we have engineered a new miRNA scaffold, miR-E, that is more efficiently processed and thus produces more potent knockdown of target genes than our previous miR30 system. By combining our sensored miR-E based shRNAs with high efficiency ES cell targeting, we have developed a fast, scalable pipeline for the production of shRNA transgenic mice with reversible gene silencing. We show that RNAi can cause sufficient knockdown to recapitulate the phenotypes of knockout mice, particularly in cancer models. More importantly, unlike traditional knockout models, RNAi has the powerful advantage of reversibility, since the endogenous gene remains intact. Using this system, we generated a number of inducible RNAi transgenic lines and demonstrate how this approach can identify predicted phenotypes and also unknown functions for well-studied genes. In addition, through regulated gene silencing we are able to mimic drug therapy in mice without the actual drug molecule, allowing us to determine the therapeutic value and/or toxic effects associated with systemic gene suppression. Using this approach, we have been able to pinpoint potential toxicities associated with gene inhibition, results that will guide drug development to avoid target failures which will likely cause harmful and intolerable effects in patients. In a model of hepatocellular carcinoma, we demonstrate that short-term inhibition of a ribosomal protein is sufficient to induce stable cell cycle arrest of liver tumor cells. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene - mice with enormous predictive power that will shape our development of better tolerated therapies.